Morphine, the gold standard for pain relief, is clinically limited by several adverse properties including tolerance, dependence and the onset of respiratory suppression. Opiate analgesics, such as morphine, mediate their biological effects mainly via activation of the mu opioid receptor (muOR). The muOR, a G protein coupled receptor (GPCR), is regulated by GPCR kinase (GRK) phosphorylation and subsequent binding of betaarrestins in a process known as receptor desensitization. The genetic ablation of betaarrestin2 (betaarr2)in mice has seemingly paradoxical effects; it leads to enhanced and prolonged morphine analgesia and dramatically reduces morphine tolerance. Moreover, these mice display no change in physical dependence; in contrast, respiratory suppression is practically eliminated. The effects of morphine in this mouse model approach a hypothetical, optimal opiate analgesic for clinical use. The coupling of the muOR to G proteins is elevated in brain regions associated with morphine analgesia in the betaarr2knockout (betaarr2-KO) mice which may contribute to the enhanced analgesic responses. However, the role of betaarr2 in morphine-induced respiratory suppression is unclear. Furthermore, while morphine analgesia is enhanced in the betaarr2-KO mice, lresponses to other opiates such as fentany and methadone, are unaltered. We have hypothesized that GRKs and betaarrestins regulate muORs and the specificity of this regulation is determined by the opiate agonist onboard. These regulatory differences at the level of the muOR may thereby underlie the diverse pharmacological effects produced by a wide-range of clinically relevant opiates such as morphine, fentanyl, methadone and buprenorphine. We have the unique opportunity to evaluate the contributions of GRKs and betarr2 to opiate responses in vivo by studying opiatemediated behaviors and physiological responses in strains of mice lacking individual GRKs (GRK2, GRK3, GRK4, GRK5, and GRK6) or betarr2. Neurochemical alterations will be assessed in these same animals in parallel to the behavioral studies with a focus on receptor trafficking, receptor desensitization, and downstream neuroadaptive changes. HEK-293 cells wil be used as a model system to further elucidate the molecular mechanisms underlying the observed in vivo phenomena. The overall objective of this study is to gain a greater understanding of muOR regulation in determining the specificity of drug effects on physiological and pathological conditions. Toward this goal, these studies focus on examining the contribution of GRKs and parrestins to muOR regulation in the development of opiate tolerance (AIM I), dependence (AIM II), and the side effects induced by opiates (AIM III). Illuminating the intricacies of muOR regulation may point to fine-tuning receptor responsiveness to increase analgesic efficacy, limit abuse liabilityand eliminate adverse side effects in developing opiate pharmaceutical therapies.